Removing an old protein fragment and/or releasing a new one has evolved as an important regulatory mechanism of many physiological processes. Research in my laboratory aims to understand the biochemical mechanism, the physiological significance, and the pathological implications of proteolysis of a group of membrane proteins, particularly the amyloid precursor protein (APR) and the Notch receptor. These proteins are first cleaved at the extracellular domains in a process called ectodomain shedding. This generates membrane-bound fragments containing new and free amino termini. Our recent work showed that the glycoprotein nicastrin binds to the new amino termini and recruits the products of ectodomain shedding for intramembrane cleavage by gamma-secretase. Recent literature and our preliminary study suggest that novel peptidase activities further process the amino or carboxyl termini of gamma-secretase products, leading to the generation of new protein fragments with biological implications. This model will now be tested in the following two hypothesis-driven Specific Aims. First, we aim to understand the functional significance and biochemical mechanism of a novel proteolytic step that modifies the amino terminus of Notch intracellular domain. Second, we will investigate the biochemical mechanism by which the carboxyl termini of amyloid-beta peptides are generated. This work will provide fundamental knowledge to proteolytic cascades of membrane proteins. It may uncover novel intracellular regulatory mechanism that could, at least partially, explain how the "stereotyped" Notch signaling pathway executes its many functions in different cellular contexts. As such, it is relevant to several Notch-associated human diseases such as Cancer, Cardiovascular disease, Stroke and Dementia. Moreover, the study will provide critical insights into the molecular nature and mode of action of the enzyme(s) responsible for generating multiple Amyloid-beta peptides in cellular membranes, and thus is relevant to understanding Alzheimer's disease.